GP13 Differential miRNA expression in CF and gene corrected iPSC-derived lung organoids

Background Cystic fibrosis (CF) is one of the most common lethal genetic diseases and is caused by mutations in the CFTR gene where a deletion of phenylalanine at position 508 (F508del) accounts for the most prevalent mutation worldwide. MicroRNA (miRNA) are small non-coding RNAs that can regulate up to two thirds of gene expression in human cells and have been shown to be dysregulated in the CF airway and implicated in the direct control of CFTR Induced Pluripotent stem cells (iPSC) can be routinely generated from any patient and contain that person’s unique genetic background, CF mutation and can form all the cell types affected by CF representing an attractive tool to advance the understanding of CF. Recent advances in iPSC technology have led to an in vitro platform of airway epithelial organoids derived from CF and CF-corrected cells for modelling CF lung disease however this model requires further molecular characterisation. We hypothesise that specific miRNA play a key role in the pathogenesis of CF lung disease and airway organoids generated from patient derived iPSCs can better elucidate this role. Methods Cystic fibrosis patient specific iPSC derived airway organoids were generated from an individual homozygous for the F508del mutation (ΔF508/ΔF508) and its gene corrected counterpart (ΔF508/wild-type [WT]) using established protocols. Expression of a miRNA panel we have previously shown to regulate CFTR (miR-145, miR-223 and miR-494) were examined in CF versus CF corrected iPSC lines at Day 0 and Day 32 following directed differentiation of iPSC into proximal airway organoids. Results In preliminary work we have observed that miRNA known to target CFTR are detectable in CF and gene-corrected iPSC lines and differential expression is observed between cells at the pluripotent stage (iPSC) at Day 0 and cells that have been differentiated in 3D as organoids to airway-like cells (bronchospheres) at Day 32. Importantly, there is differential expression of key CFTR related miRNA when we compare CF patient-derived bronchospheres to those where the F508del mutation has been corrected. Increased expression of miR-494 was observed in CF vs. CF corrected cells whereas levels of miR-145 and -223 appeared to be reduced. Conclusions We have demonstrated for the first time that miRNA are detectable in CF iPSC derived bronchospheres with altered expression following a directed differentiation approach of iPSC into proximalised airway epithelium. Expression patterns in iPSC may be different to patient bronchial epithelial cells previously described and warrants further investigation.